A Novel Method for Analyzing Mitochondrial Movement: Inhibition by Paclitaxel in a Pheochromocytoma Cell Model

Abstract

A method was developed to assess mitochondrial movement in the living cell that is dependent, in part, on microtubule and/or associating protein interactions. The leader sequence from cytochrome-c was used to drive DsRed2 fluorescent proteins to accumulate in the mitochondria, thus enabling to follow mitochondrial (cytochrome-c's) movement. For calculating the percentage of mitochondrial movement, an image-processing program was used (ImageJ). Paclitaxel, an antitumor agent, is a potent microtubule-stabilizing agent that increases the stability of tubulin polymers, inhibiting mitosis and mitochondrial activity in dividing cells. Here, we tested whether paclitaxel inhibits mitochondrial movement in pheochromocytoma cells (a neuronal model, when tested in a differentiated state). While a 2-day exposure to paclitaxel resulted in cellular toxicity (measured as inhibition of mitochondrial activity), 2-3 h exposure to paclitaxel were sufficient to inhibit mitochondrial movement as assessed in 10-20-s imaging sessions in living cells. Mitotracker deep-red staining validated the staining obtained with DsRed2-cytochrome-c and identified intact mitochondria. Results showed a significant paclitaxel dose-dependent inhibition of mitochondrial movement. This new method should enable further assessment of microtubule-interacting drugs and other cytoskeletal components for their potential influence of mitochondrial movement as a test for activity and side effects.

Paclitaxel Treatment and Proprotein Convertase 1/3 (PC1/3) Knockdown in Macrophages is a Promising Antiglioma Strategy as Revealed by Proteomics and Cytotoxicity Studies

“…We found that in both NT and PC1/3 KD cells, the Paclitaxelinduced up-regulation of Tuba1ac and Tuba4a was accompanied by the co-upregulation of a highly significant number of mitochondria-related molecules (Figure 3Ci). This result was in line with previous studies that demonstrated that Paclitaxel targets mitochondria 33 , possibly as a direct consequence of microtubule network alterations 34 . Interestingly, we also observed that Tuba1ac/Tuba4a co-expression networks comprised immune-related molecules that, for some, were distinct when comparing Paclitaxel-treated NT cells vs. Paclitaxel-treated PC1/3 KD cells.…”

Mitochondrial respiratory dysfunction‐elicited oxidative stress and posttranslational protein modification in mitochondrial diseases

“…A recent study showed that paclitaxel, a microtubule-stabilizing drug, could impair the function and motility of mitochondria through enhancing the stability of tubulin polymers. 35 These findings support our contention that mitochondrial defect may induce acetylation and detyrosination of ␣-tubulin to increase mirotubule rigidity, which in turn contributes to the alteration of mitochondrial network in the skin fibroblasts of CPEO patients. In a further study, we showed that a decrease in the expression of tubulin deacetylase Sirt2 was associated with the accumulation of acetylated ␣-tubulins in the skin fibroblasts of CPEO patients (Fig.…”

Nerve growth factor alters microtubule targeting agent-induced neurotransmitter release but not MTA-induced neurite retraction in sensory neurons

“…Varied hypotheses have been proposed, including: compromised transport of proteins and organelles (Shprung and Gozes, 2009, Shemesh and Spira, 2010, LaPointe et al, 2013), changes in receptor and channel localization and function (Flatters and Bennett, 2004, Matsumoto et al, 2006, Xiao et al, 2007, Nieto et al, 2008, Chen et al, 2011, Okubo et al, 2011, Kawakami et al, 2012, Hara et al, 2013, Chen et al, 2014) and/or activation of an immune response (Siau et al, 2006, Peters et al, 2007, Pevida et al, 2013, Warwick and Hanani, 2013, Zhang et al, 2013, Li et al, 2014, Li et al, 2015). Stabilization of microtubules can alter both anterograde and retrograde axonal transport of mitochondria along the microtubules (Nennesmo and Reinholt, 1988, Morris and Hollenbeck, 1995, Nakata and Yorifuji, 1999, Theiss and Meller, 2000, Shemesh and Spira, 2010, Bober et al, 2015).…”